Inhibitors of fatty acid uptake and methods of use

ABSTRACT

The present disclosure describes inhibitors of fatty acid uptake and methods of using such inhibitors. Specifically, the present disclosure describes inhibitors with specificity for FATP2.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. 119(e) to U.S.Application No. 61/217,498, filed on Jun. 1, 2009.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. D071076awarded by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

This disclosure generally relates to inhibitors of fatty acid uptake.

BACKGROUND

It is becoming increasingly apparent that typical western diets high infat lead to a plethora of pathophysiological states ranging from obesityand type 2 diabetes to coronary heart disease. The correlation betweenchronically elevated plasma free fatty acids and triglycerides with thedevelopment of obesity, insulin resistance and cardiovascular diseasehas led to the hypothesis that decreases in pancreatic insulinproduction, cardiac failure, arrhythmias, and hypertrophy are due toaberrant accumulation of lipids in these tissues that normally do notstore significant levels of fatty acids and triglycerides.

SUMMARY

The present disclosure describes inhibitors of fatty acid uptake andmethods of using such inhibitors.

Described herein is a method of inhibiting fatty acid uptake by cells,such as intestinal epithelial cells and hepatocytes. The method caninclude contacting the cells with an inhibitor as provided herein. Inone embodiment, the inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt form thereof, wherein:

-   -   X is O or S;    -   R¹ is selected from the group consisting of: F, Cl, Br, I, OH,        and O(C₁₋₆ alkyl);    -   R², R³, and R⁴ are independently selected from the group        consisting of: H, F, Cl, Br, I, OH, and O(C₁₋₆ alkyl), wherein        at least two of R², R³, and R⁴ are not H.

In some embodiments, X is S. In some embodiments, R¹ is selected fromthe group consisting of: F, Cl, and O(C₁₋₆ alkyl). In some embodiments,R² is selected from H and Br. In some embodiments, R³ is OH. In someembodiments, R⁴ is O(C₁₋₆ alkyl).

Non-limiting examples of a compound of Formula (I) include:

or a pharmaceutically acceptable salt form thereof.

In another embodiment, an inhibitor as described herein is a compound ofFormula (II):

or a pharmaceutically acceptable salt form thereof, wherein:

-   -   R¹ is H or NO₂;    -   R² is selected from the group consisting of: H, F, Cl, Br, I,        OH, and O(C₁₋₆ alkyl);    -   R³ is selected from the group consisting of: F, Cl, Br, I, OH,        and O(C₁₋₆ alkyl); and    -   n is an integer from 0 to 5.

In some embodiments, R¹ is NO₂. In some embodiments, R² is selected fromthe group consisting of: H, Cl, and O(C₁₋₆ alkyl). In some embodiments,n is 0.

Non-limiting examples of a compound of Formula (II) include:

or a pharmaceutically acceptable salt form thereof.

In some embodiments, an inhibitor as provided herein is a compound ofFormula (III):

or a pharmaceutically acceptable salt form thereof, wherein:

-   -   R¹ is H or O(C₁₋₆ alkyl); and    -   R² is a substituted or unsubstituted heterocycloalkyl or        heteroaryl.

In some embodiments, R¹ is H. In some embodiments, R² is a substitutedor unsubstituted heterocylcoalkyl.

Non-limiting examples of a compound of Formula (III) include:

or a pharmaceutically acceptable salt form thereof.

Further provided herein are inhibitors selected from a compound ofFormula (IV):

or a pharmaceutically acceptable salt form thereof, wherein:

-   -   R¹ and R² are independently selected from the group consisting        of: H, F, Cl, Br, I, OH, and O(C₁₋₆ alkyl).

In some embodiments, R¹ is selected from H and OH. In some embodiments,R² is selected from H and Br.

Non-limiting examples of a compound of Formula (IV) includes:

or a pharmaceutically acceptable salt form thereof.

In some embodiments, an inhibitor as provided herein is a compound:

or a pharmaceutically acceptable salt form thereof.

Also provided herein is a method of treating a disease, comprising:administering, to an individual, an inhibitor as described herein. Insome embodiments, the disease being treated can include obesity,metabolic syndrome, insulin resistant diabetes, cardiovascular disease,stroke, gallbladder disease, osteoarthritis, sleep apnea, respiratoryproblems and cancer.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods and compositions of matter belong. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the methods and compositionsof matter, suitable methods and materials are described below. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety.

DESCRIPTION OF DRAWINGS

FIG. 1 shows that the titration of selected compounds yields sigmoidaldose-response curves for the inhibition of C₁-BODIPY-C₁₂ uptake intoCaco-2 cells. Curves were fit using dose-response nonlinearleast-squares regression models in Prism software. (A) compound I-1; (B)compound II-1; (C) compound III-1; (D) compound IV-1; and (E) compoundV-1. Values are presented as means±SE for 3-4 experiments assayed intriplicate.

FIG. 2 shows the dose-response curves for the inhibition ofC₁-BODIPY-C₁₂ uptake into 3T3-L1 adipocytes. Curves were fit usingdose-response nonlinear least-squares regression models in Prismsoftware. (A) compound I-1; (B) compound II-1; (C) compound III-1; (D)compound IV-1; and (E) compound V-1. Values are presented as means±SEfor 4 experiments assayed in triplicate.

FIG. 3 shows that the selected compounds are not toxic to cells. Caco-2cells were treated with I-1, II-1, III-1 or IV-1 at 50 μM, or V-1 at 100μM for 24 h, then the MTT assay was performed to assess mitochondrialfunction, an indicator of cellular toxicity. Bar height indicates themean value±standard deviation for 2 experiments assayed in triplicate.

FIG. 4 shows the evaluation of barrier and membrane function of Caco-2cells after compound treatment. (A) Trans-epithelial electricalresistance (TEER) was measured in fully differentiated Caco-2 cellsafter one hour treatment with two different concentrations of selectedcompounds as shown. Caco-2 cells were seeded and differentiated inCollagen-Coated Transwell®-COL Inserts. TEER was measured using aMillipore Millicell®-ERS device. (B) Transport of the glucose analogue2-NBDG was measured in Caco-2 cells seeded and differentiated in 96-wellplates. Final concentrations of compounds were 20 μM for compounds I-1,II-1, III-1 and IV-1; and 50 μM for compound V-1. Bar height indicatesmean values±standard deviation for 3-5 independent experiments assayedin duplicate (A) and triplicate (B), respectively.

FIG. 5 shows the inhibition of uptake of [³H]C_(18:1) into Caco-2 cells.Caco-2 cells were preincubated for one hour with selected compounds(final concentrations were 20 μM for compounds I-1, II-1, III-1 andIV-1; and 50 μM for compound V-1) followed by the addition of[³H]C_(18:1) for 3 minutes as detailed in the text. Bar height indicatesthe mean values±standard deviation for 3-5 independent experiments.

FIG. 6 shows the assessment of oleoyl (C_(18:1))-CoA synthetase activityafter compound treatment. Cells were treated for 1 h at the indicatedfinal concentration, then cell extracts were prepared and Acsl activitymeasured. Bar height indicates mean values±standard deviation for 3-5independent experiments assayed in duplicate.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

Fatty acids are enigmatic molecules that, on the one hand, are essentialfor cellular structure, function and signaling and, on the other hand,must be contained or their detergent properties will prove lethal tocells. Mother Nature has, therefore, developed ways to compartmentalize,sequester and regulate the movement of these molecules between andwithin cells. For example, within the blood stream, free fatty acids(FFA) are buffered and moved by serum albumin, and as complex lipids bythe lipoproteins. Within cells, the fatty acid binding proteins serve asimilar function for the free carboxylic acids, while fatty acidsesterified in highly hydrophobic complex lipid species are partitionedinto membranes or sequestered in lipid droplets.

Upon presentation to the cell, fatty acids must be transported acrossthe cell membrane and trafficked to sites of utilization. Generally, thefree fatty acid concentration in the extracellular space is extremelylow. Therefore, the efficient transport of long-chain fatty acids isexpected to require specific membrane-bound and membrane-associatedtransport systems to accumulate these compounds against a concentrationgradient, where the intracellular concentrations of fatty acids are upto two- to three-fold higher than external unbound fatty acidconcentrations. Different cell types contain a specific repertoire ofmembrane-bound and membrane-associated proteins, which are believed togovern fatty acid transport in response to differentiation, hormonalstimulus, or environmental stimulus, including changes in nutritionalstate, temperature, or oxygen availability.

Specific membrane-bound and membrane-associated proteins have beenidentified that function in one or more steps in the transport of fattyacids across the membrane. To date, four different membrane-bound ormembrane associated proteins have been defined in eukaryotic cells thatparticipate in the transport of exogenous long-chain fatty acids:CD36/fatty acid translocase (CD36/FAT), fatty acid bindingprotein—plasma membrane-bound (FABPpm), fatty acid transport protein(FATP), and long chain acyl CoA synthetase (Acsl). One of theprotein-mediated processes by which fatty acid transport occurs isthrough vectorial acylation, which involves specific FATP isoforms thatfunction alone or in concert with a long chain acyl-CoA synthetase(Acsl). In this coupled transport mechanism, the exogenous fatty acid isactivated by esterification with coenzyme A concomitant with transport.

Fatty acid transport proteins (FATPs) were first identified infunctional cloning screens designed to identify proteins that resultedin elevated accumulation of fatty acids. FATPs have a domainarchitecture similar to the acyl-CoA synthetases, which includes an ATPbinding domain and a fatty acid binding domain. Generally, however, thefatty acid signature of FATPs is divergent from the Acsl enzymesinvolved in activation of long chain fatty acids. In addition to theFATP1, five other related mammalian genes, referred to as FATP2 throughFATP6, have been cloned and their proteins characterized. The FATPsdiffer in expression pattern, tissue distribution and subcellularlocation. For example, FATP1 is found in muscle and adipose tissue;FATP2 in liver and kidney; FATP3 in liver and testes; FATP4 isrelatively ubiquitous in fat metabolizing tissues and skin; FATP5 isexclusive to liver; and FATP6 is exclusive to heart. Each FATP functionsin thioesterification of a lipophilic substrate with coenzyme A. FATP2,the target of the screen described herein, was first identified as avery long chain acyl-CoA synthetase, called ACSVL1.

This disclosure describes inhibitors of FATP2, which representcandidates for therapeutic applications involved in inhibiting fattyacid transport mediated through FATP2.

Inhibitors of Fatty Acid Transport Proteins

The inhibitors described herein were identified in a screen using a highthroughput screening assay (see, U.S. Pat. No. 7,070,944). Theinhibitors identified could be categorized into 5 classes of compoundsas shown below. The inhibitory properties of each of these 5 classes ofcompounds was reversible, none disrupted the barrier function ofepithelial cells, and each specifically blocked fatty acid transportwithout disrupting glucose transport or perturbing cell integrity.Interestingly, these compounds were less effective in blocking fattyacid transport in adipocytes, which express FATP1.

A compound of Formula (I):

or a pharmaceutically acceptable salt form thereof, wherein:

-   -   X is O or S;    -   R¹ is selected from the group consisting of: F, Cl, Br, I, OH,        and O(C₁₋₆ alkyl);    -   R², R³, and R⁴ are independently selected from the group        consisting of: H, F, Cl, Br, I, OH, and O(C₁₋₆ alkyl), wherein        at least two of R², R³, and R⁴ are not H.

In some embodiments, X is S. In some embodiments, R¹ is selected fromthe group selected from: F, Cl, and O(C₁₋₆ alkyl). In some embodiments,R¹ is OCH₃. In some embodiments R² is selected from H and Br. In someembodiments, R³ is OH. In some embodiments, R⁴ is O(C₁₋₆ alkyl). In someembodiments, if R¹ is in the para position and is OCH₃ and X is S, thenR², R³, and R⁴ are not H, OH, and OH, respectively. In some embodiments,if R¹ is in the meta position and is Cl and X is O, then R², R³, and R⁴are not Br, OH, and OCH₃, respectively.

Non-limiting examples of compounds according to Formula (I) include:

or a pharmaceutically acceptable salt form thereof.

A compound of Formula (II):

or a pharmaceutically acceptable salt form thereof, wherein:

-   -   R¹ is H or NO₂;    -   R² is selected from the group consisting of: H, F, Cl, Br, I,        OH, and O(C₁₋₆ alkyl);    -   R³ is selected from the group consisting of: F, Cl, Br, I, OH,        and O(C₁₋₆ alkyl); and    -   n is an integer from 0 to 5.

In some embodiments, R¹ is NO₂. In some embodiments, R² is selected fromthe group consisting of: H, Cl, and O(C₁₋₆ alkyl). In some embodiments,R² is OCH₃. In some embodiments, n is 0.

In some embodiments, a compound of Formula (II) is a compound accordingto Formula (IIA):

or a pharmaceutically acceptable salt form thereof, wherein:

-   -   R¹ is H or NO₂;    -   R² is selected from the group consisting of: H, F, Cl, Br, I,        OH, and O(C₁₋₆ alkyl).

In some embodiments, R¹ is NO₂. In some embodiments, R² is selected fromthe group consisting of: H, Cl, and O(C₁₋₆ alkyl). In some embodiments,R² is OCH₃.

Non-limiting examples of compounds according to Formula (II) and Formula(IIA) include:

or a pharmaceutically acceptable salt form thereof.

A compound according for Formula (III):

or a pharmaceutically acceptable salt form thereof, wherein:

-   -   R¹ is H or O(C₁₋₆ alkyl); and    -   R² is a substituted or unsubstituted heterocycloalkyl or        heteroaryl.

In some embodiments, R¹ is H. In some embodiments, R² is a substitutedor unsubstituted heterocycloalkyl. In some embodiments, R² is asubstituted or unsubstituted dihydrodiazapine. In some embodiments, ifR² is a substituted pyrazole, R¹ is not H.

A non-limiting example of a compound according to Formula (III)includes:

or a pharmaceutically acceptable salt form thereof.

A compound according to Formula (IV):

or a pharmaceutically acceptable salt form thereof, wherein:

-   -   R¹ and R² are independently selected from the group consisting        of: H, F, Cl, Br, I, OH, and O(C₁₋₆ alkyl).

In some embodiments, R¹ is selected from OH and H. In some embodiments,R² is selected from H and Br.

Non-limiting examples of compounds according to Formula (IV) include:

or a pharmaceutically acceptable salt form thereof.

A further example of a compound as described herein includes:

or a pharmaceutically acceptable salt form thereof.

The term “alkyl” includes straight-chain alkyl groups (e.g., methyl,ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.)and branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.).cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In certain embodiments, a straightchain or branched chain alkyl has 6 or fewer carbon atoms in itsbackbone (e.g., C₁₋₆ for straight chain, C₃₋₆ for branched chain). Theterm C₁₋₆ includes alkyl groups containing 1 to 6 carbon atoms.

The term “heteroaryl” includes groups, including 5- and 6-memberedsingle-ring aromatic groups, that have from one to four heteroatoms, forexample, pyrrole, furan, dihydrodiazapine, thiophene, thiazole,isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “heteroaryl” includes multicyclic heteroarylgroups, e.g., tricyclic, bicyclic, such as benzoxazole, benzodioxazole,benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl,quinoline, isoquinoline, napthyridine, indole, benzofuran, purine,benzofuran, quinazoline, deazapurine, indazole, or indolizine.

The term “heterocycloalkyl” includes groups, including but not limitedto, 3- to 10-membered single or multiple rings having one to fiveheteroatoms, for example, piperazine, pyrrolidine, piperidine, orhomopiperazine.

The term “substituted” means that an atom or group of atoms formallyreplaces hydrogen as a “substituent” attached to another group. Forheteroaryl and heterocycloalkyl groups, the term “substituted”, unlessotherwise indicated, refers to any level of substitution, namely mono,di, tri, tetra, or penta substitution, where such substitution ispermitted. The substituents are independently selected, and substitutionmay be at any chemically accessible position.

Pharmaceutically acceptable salts of the compounds described hereininclude the acid addition and base salts thereof.

Suitable acid addition salts are formed from acids which form non-toxicsalts. Examples include the acetate, adipate, aspartate, benzoate,besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate,citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate,gluconate, glucuronate, hexafluorophosphate, hibenzate,hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide,hydrogen phosphate, isethionate, D- and L-lactate, malate, maleate,malonate, mesylate, methylsulphate, 2-napsylate, nicotinate, nitrate,orotate, oxalate, palmitate, pamoate, phosphate/hydrogen,phosphate/phosphate dihydrogen, pyroglutamate, saccharate, stearate,succinate, tannate, D- and L-tartrate, 1-hydroxy-2-naphthoate tosylateand xinafoate salts.

Suitable base salts are formed from bases which form non-toxic salts.Examples include the aluminium, arginine, benzathine, calcium, choline,diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine,potassium, sodium, tromethamine and zinc salts.

Hemisalts of acids and bases may also be formed, for example,hemisulphate and hemicalcium salts.

A person skilled in the art will know how to prepare and select suitablesalt forms for example, as described in “Handbook of PharmaceuticalSalts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH,Weinheim, Germany, 2002).

The compounds for use in the compositions and methods provided hereinmay be obtained from commercial sources (e.g., Aldrich Chemical Co.,Milwaukee, Wis.) or may be prepared by methods well known to those ofskill in the art, e.g., by those methods discussed in standard textbookssuch as “Comprehensive Organic Transformations—A Guide to FunctionalGroup Transformations”, R C Larock, Wiley-VCH (1999 or later editions).One of skill in the art would be able to prepare all of the compoundsfor use herein by routine modification of these methods using theappropriate starting materials.

Methods of Using Inhibitors of Fatty Acid Transport Proteins

The five classes of inhibitor compounds described herein can be used toinhibit fatty acid uptake by cells. Such inhibitor compounds can be usedto prevent fatty acid transport by cells as a method to limit dietaryfat absorption into tissues and organs that are susceptible to the toxiceffects of excessive fatty acids (e.g., pancreas, liver and muscle).Thus, these inhibitors are candidates for combating obesity andchronically high blood triglycerides and/or free fatty acids, and can beused to treat a number of diseases associated with dyslipidemias andlipotoxicity in humans or companion animals including, for example,metabolic syndrome, insulin resistant diabetes, cardiovascular disease,stroke, gallbladder disease, non-alcoholic fatty liver disease,osteoarthritis, sleep apnea, respiratory problems and certain cancers(e.g., endometrial, breast and colon).

The compounds described herein intended for pharmaceutical use may beadministered as crystalline or amorphous products. They may be obtained,for example, as solid plugs, powders, or films by methods such asprecipitation, crystallization, freeze drying, spray drying, orevaporative drying. Microwave or radio frequency drying may be used forthis purpose.

They may be administered alone or in combination with one or more othercompounds described herein or in combination with one or more otherdrugs (or as any combination thereof). Generally, they will beadministered as a formulation in association with one or morepharmaceutically acceptable excipients. The term “excipient” is usedherein to describe any ingredient other than the compound(s) of theinvention. The choice of excipient will to a large extent depend onfactors such as the particular mode of administration, the effect of theexcipient on solubility and stability, and the nature of the dosageform.

Non-limiting examples of pharmaceutical excipients suitable foradministration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration. Pharmaceutically acceptableexcipients include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, self-emulsifying drug delivery systems(SEDDS) such as d-α-tocopherol polyethylene glycol 1000 succinate,surfactants used in pharmaceutical dosage forms such as Tweens or othersimilar polymeric delivery matrices, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate,sodium-chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethyl cellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, and wool fat.Cyclodextrins such as α-, β, and γ-cyclodextrin, or chemically modifiedderivatives such as hydroxyalkylcyclodextrins, including 2- and3-hydroxypropyl-b-cyclodextrins, or other solubilized derivatives canalso be advantageously used to enhance delivery of compounds of theformulae described herein. In some embodiments, the excipient is aphysiologically acceptable saline solution.

The compositions can be, in one embodiment, formulated into suitablepharmaceutical preparations such as solutions, suspensions, tablets,dispersible tablets, pills, capsules, powders, sustained releaseformulations or elixirs, for oral administration or in sterile solutionsor suspensions for parenteral administration, as well as transdermalpatch preparation and dry powder inhalers (see, e.g., Ansel Introductionto Pharmaceutical Dosage Forms, Fourth Edition 1985, 126).

The concentration of a compound in a pharmaceutical composition willdepend on absorption, inactivation and excretion rates of the compound,the physicochemical characteristics of the compound, the dosageschedule, and amount administered as well as other factors known tothose of skill in the art.

The pharmaceutical composition may be administered at once, or may bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration oftreatment is a function of the disease being treated and may bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular patient, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed compositions.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the compounds or pharmaceutically acceptablederivatives thereof. The pharmaceutically therapeutically activecompounds and derivatives thereof are, in one embodiment, formulated andadministered in unit-dosage forms or multiple-dosage forms. Unit-doseforms, as used herein, refers to physically discrete units suitable forhuman and animal patients and packaged individually as is known in theart. Each unit-dose contains a predetermined quantity of thetherapeutically active compound sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier, vehicle or diluent. Examples of unit-dose forms includeampoules and syringes and individually packaged tablets or capsules.Unit-dose forms may be administered in fractions or multiples thereof. Amultiple-dose form is a plurality of identical unit-dosage formspackaged in a single container to be administered in segregatedunit-dose form. Examples of multiple-dose forms include vials, bottlesof tablets or capsules or bottles of pints or gallons. Hence, multipledose form is a multiple of unit-doses which are not segregated inpackaging.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing an activecompound as defined above and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, solubilizingagents, pH buffering agents and the like, for example, acetate, sodiumcitrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and other suchagents.

Dosage forms or compositions containing a compound as described hereinin the range of 0.005% to 100% with the balance made up from non-toxiccarrier may be prepared. Methods for preparation of these compositionsare known to those skilled in the art. The contemplated compositions maycontain 0.001%-100% active ingredient, in one embodiment 0.1-95%, inanother embodiment 75-85%.

Pharmaceutical compositions suitable for the delivery of compoundsdescribed herein and methods for their preparation will be readilyapparent to those skilled in the art. Such compositions and methods fortheir preparation may be found, for example, in “Remington'sPharmaceutical Sciences”, 19th Edition (Mack Publishing Company, 1995).

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, biochemical, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. The invention will be furtherdescribed in the following examples, which do not limit the scope of themethods and compositions of matter described in the claims.

EXAMPLES Example 1 Materials

Yeast extract, yeast peptone, yeast nitrogen base without amino acids,and dextrose were obtained from Difco (Detroit, Mich.). Complete aminoacid supplement and individual amino acids were obtained from Sigma (St.Louis, Mo.). Fatty-acid-free bovine serum albumin (FAF BSA) and otherchemical reagents were also obtained from Sigma. The fluorescentlong-chain fatty acid analog,4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid(C₁-BODIPY-C₁₂) and the fluorescent glucose analog,2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG)were purchased from Molecular Probes/Invitrogen (Eugene, Oreg.).Radioactive fatty acid ([³H] oleate; specific activity, 5 mCi/ml) wasobtained from Perkin Elmer (Waltham, Mass.). BD Biosciences black withclear bottom 384-well and 96-well microplates were used for primary andsecondary screenings in yeast. For Caco-2 and HepG2 cells, tissueculture-treated 96-well black with clear flat bottom polystyrenecollagen-coated microplates were obtained from Fisher Scientific(Pittsburgh, Pa.). Transwell® Permeable Supports used for experimentsinvolving Caco-2 cells were obtained from Corning Life Sciences (Acton,Mass.).

Example 2 Cell Culture and Reagents

S. cerevisiae strain LS2086 containing deletions within the FAT1 andFAA1 genes (fat1Δfaa1Δ; MATa ura3 52 his3200 ade2-101 lys2-801leu2-3,112 faa1Δ:HIS3 fat1Δ::G418) expressing hsFATP2 was used for theprimary high throughput screening as detailed previously [Li et al., J.Lipid Res., 2008, 49:230-44; Zou et al., J. Biol. Chem., 2003,278:16414-22]. For most experiments, yeast minimal medium with dextrose(YNBD) containing 0.67% yeast nitrogen base (YNB), 2% dextrose, adenine(20 mg/L), uracil (20 mg/L), and amino acids as required [arginine,tryptophan, methionine, histidine, and tyrosine (20 mg/L); lysine (30mg/L); and leucine (100 mg/L)] was used. When a rich medium wasrequired, yeast complete media with adenine (YPDA) was used. Growth inliquid culture and on plates was at 30° C.

The Caco-2 cell line is derived from a human adenocarcinoma and is ableto undergo differentiation into polarized epithelial cells that show abrush border phenotype and form well-developed and functional tightjunction complexes [Fogh et al., J. Natl. Cancer Inst., 1977, 59:221-6;Grasset et al., Am. J. Physiol., 1984, 247:C260-7]. This cell line isused as an in vitro model to predict human intestinal absorption andsecretion [Hilgers et al., Pharm. Res., 1990, 7:902-10]. Caco-2 cellswere maintained in Earl's minimal essential medium (MEM) with 20% FBS ina 95% air 5% CO₂ atmosphere at 37° C., as previously described [Sandovalet al., Arch. Biochem. Biophys., 2008, 477:363-71]. For growth anddifferentiation, the BD Biosciences Intestinal EpitheliumDifferentiation Media Pack was used. Cells were plated in basal seedingmedium at a density of 2.5×10⁵ cells/cm² on a collagen-coatedblack-clear 96-well plate (BD Biosciences). After 72 h in culture, thebasal seeding medium was removed and Entero-STIM medium was added toeach well. Both media contained mito-serum extender. After another 24 h,cells were serum-starved for one hour in MEM without phenol red prior toperforming the C₁-BODIPY-C₁₂ uptake assay. HepG2 cells (ATCC, HB-8065)were cultured according to the ATCC protocol. The cells were seeded in96-well collagen coated plates at a seeding density of 2.5×10⁵cells/cm². 3T3-L1 fibroblasts (ATCC, CL-173) were maintained in modifiedDMEM and 10% BCS. For differentiation into adipocytes, 3T3-L1 cells weretreated with methylisobutylxanthine (0.5 mM), dexamethasone (1.0 μM),and insulin (1.75 μM) in DMEM and 10% FBS for 48 hours as described byStudent et al. [Student et al., J. Biol. Chem., 1980, 255:4745-50].After 48 hour incubation with differentiation medium, cells were treatedwith DMEM and 10% FBS supplemented with insulin (1.75 μM) for 48 hours.Cells were subsequently maintained in DMEM and 10% FBS for an additional3-6 days until fully differentiated. The number of cells per well inscreening trials were determined using the FluoReporter® BlueFluorometric DNA Quantitation Kit from Invitrogen.

Example 3 Library Descriptions and High Throughput Screening

Two chemically diverse compound libraries were screened using a recentlydeveloped live-cell HTS method [Li et al., J. Lipid. Res., 2008,49:230-44]. The NCI Diversity Set Compound Library comprises 1990chemical core structures representative of a larger compound library of140,000 compounds (obtained from NCI's Developmental TherapeuticsProgram (DTP)). The ChemBridge Corporation compound library includes adiverse, drug-like collection of 100,000 compounds.

For primary screening, LS2086 transformed with the hsFATP2 expressionvector (pDB126) or transformed with the empty vector (pDB121) along withthe GAL4 transcription factor fusion vector, pRS416Gal4-ER-VP16,[Stafford & Morse, J. Biol. Chem., 1997, 272:11526-34] were pre-grown inYNBD without leucine and uracil (YNBD-leu-ura) as described [Li et al.,J. Lipid Res., 2008, 49:230-44]. The cells were subsequently subculturedto A_(600 nm) of 0.02 in the same medium containing 10 nM β-estradiol toinduce FATP2 expression, grown to mid-log-phase (0.8-1.2 A₆₀₀),harvested and then resuspended in PBS to a final density of 6×10⁷cells/ml prior to dispensing into a 384-well assay plate (22.5 μ/well;(1.35×10⁶ cells)). Wells in the first two rows of each 384-well platereceived the vector control cells, and all other wells in the platereceived cells expressing hsFATP2. Compounds (2.5 μL) were then added toa final concentration of 40 μM in PBS. After a 2 hr incubation at 30°C., 75 μL of the C₁-BODIPY-C₁₂ transport mixture (resulting in finalconcentrations 1.25 μM C₁-BODIPY-C₁₂, 0.75 μM FAF BSA, 2.1 mM Trypanblue) were added to each well. After 30 min, the cell-associatedfluorescence, reflective of fatty acid transport, was measured using aBio-Tek Synergy HT multidetection microplate reader (Bio-TekInstruments, Inc. Winooski, Vt.) using filter sets of 485 nm±20excitation and 528 nm ±20 emission. The Z′ factor for each plate wascalculated using cells expressing hsFATP and vector controls withoutcompound essentially as described [Li et al., J. Lipid Res., 2008,49:230-44]. A compound was considered a primary hit when the finalfluorescence value was three standard deviation units above or below thepositive control fluorescence value obtained for each plate assayed. Allcompounds that resulted in an increase in cell-associated fluorescencewere found to be autofluorescent and were not considered further.

Secondary screens were performed to identify compounds that actednon-specifically as described previously [Li et al., J. Lipid Res.,2008, 49:230-44]. Compounds were eliminated from further considerationif (a) they were autofluorescent, (b) were able to quench the BODIPYfluorophore, or (c) permeabilized the cells to allow internalization andquenching of trypan blue. Compounds passing each of these secondaryscreens were subsequently tested for activity primarily using Caco-2cells as detailed below.

Example 4 Compound Evaluation in Caco-2 Cells, HepG2 Cells and 3T3-L1Adipocytes

Caco-2 cells were plated in basal seeding medium at a density of 2.5×10⁵cells/cm² in collagen-coated 96-well plates, and differentiated asdetailed above. HepG2 cells were cultured in EMEM. Differentiated 3T3-L1adipocytes were maintained in modified DMEM and 10% FBS. After another24 h, cells were serum-starved for 1 h in MEM without phenol red priorto performing the C₁-BODIPY-C₁₂ transport assay [Sandoval et al., Arch.Biochem. Biophys., 2008, 477:363-71; Arias-Barrau et al., Methods Mol.Biol., 2009, 580:233-49]. In a standard reaction, serum-free MEM wasremoved from the wells and 50 μL of the test compound in MEM (MEM alonefor controls) were added to each well and incubation was continued for 1h. Then 50 μL of C₁-BODIPY-C₁₂ mixture (final concentrations 5 μMC₁-BODIPY-C₁₂; 5 μM FAF BSA; 1.97 mM trypan blue) was added to eachwell, and uptake was allowed to take place for 15 min. Cell-associatedfluorescence was measured as detailed above. The inhibition of fattyacid uptake activity using C₁-BODIPY-C₁₂ was assayed using differentconcentrations of selected compounds ranging from 0.001 μM to 640 μM.Ligand competition curves were fit by nonlinear least-squares regressionusing one-site competition and dose-response models and Prism software(GraphPad software, Inc., San Diego, Calif.) in order to determine thecompound concentration that reduced C₁-BODIPY-C₁₂ fluorescence readoutby 50% (IC₅₀). K_(i) values were calculated from the IC₅₀ using theequation of Cheng and Prusoff as detailed in Li et al. [J. Lipid Res.,2008, 49:230-44] and K_(T) values published by Sandoval et al. [Sandovalet al., Arch. Biochem. Biophys., 2008, 477:363-71].

To evaluate if inhibition of fatty acid uptake after compound treatmentwas reversible, cells were seeded in 96-well plates and treated asdescribed above, but after 1 h the media containing the compound wasremoved, cells were washed twice with MEM, and fresh media containingserum was added. Cells were incubated 24 h at 37° C. with 5% CO₂ andthen fatty acid uptake was measured using the standard C₁-BODIPY-C₁₂transport assay.

Example 5 Cytotoxicity Assay in Caco-2 Cells

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]assay was used to determine if compounds of interest were cytotoxic toCaco-2 cells [Mosmann, J. Immunol. Methods, 1983, 65:55-63]. Cells werecultured and differentiated as detailed above in collagen-coated 96-wellplates. Cells were incubated at 37° C., 5% CO₂ for at least one hour andup to 72 h in MEM containing the appropriate dilution of compound.Following this incubation period, the media with compound was removedand 110 μL of MTT reagent (prepared in MEM (final concentration 0.45mg/mL MTT)) was added. After a 3 h incubation period, the reaction wasterminated by the addition of 150 μL stop buffer (0.01 N HCl in 10%SDS). The plates were incubated for 1 h at 37° C. to facilitatesolubilization of formazan crystals; color development was read at A₅₇₀.

Example 6 Long Chain Acyl-CoA Synthetase (Acsl) Activity in Caco-2 Cellsafter Compound Treatment

Caco-2 cells were grown and differentiated in 60 mm collagen coateddishes (at a seeding density 2.5×10⁵ cells/cm²). Following growth anddifferentiation as detailed above, cells were serum starved for 1 h inMEM and then were treated for 1 h with selected compounds at specifiedfinal concentrations. The media was subsequently removed by aspirationand cells washed once with 5 mL PBS, trypsinized using standardprocedures and collected by centrifugation. The cell pellet wasresuspended in 1 mL STE lysis buffer (10 mM Tris-HCl (pH 8.0); 0.1 MNaCl; 1 mM EDTA) and sonicated on ice for 2 min using 3 sec on/offpulses. Samples were clarified by centrifugation (15,000×g, 15 min, 4°C.), the supernatant was transferred to a new tube and assayed foroleoyl-CoA synthetase activity (see below); protein concentrations weredetermined using the Bradford assay with bovine serum albumin as astandard [Bradford, Analytical Biochemistry, 1976, 72:248-54]. Inparallel experiments, untreated cells were used to prepare the cellextract for Acsl activity measurements where the compound of interestwas added directly to the reaction mixture.

Oleoyl-CoA synthetase activity was determined using a reaction mixturecontaining 200 mM Tris-HCl (pH 7.5), 2.5 mM ATP, 8 mM MgCl₂, 2 mM EDTA,20 mM NaF, 0.01% Triton X-100, 50 μM [³H]-oleic acid (C_(18:1))(dissolved in 10 mg/mL α-cyclodextrin), and 0.5 mM coenzyme A.Individual enzyme reactions were initiated by the addition of coenzymeA, incubated at 30° C. for 20 min, and terminated by the addition of 2.5mL of Dole's Reagent (isopropyl alcohol, n-heptane, 1 M H₂SO₄ (40:10:1)[Dole, J. Clin. Invest., 1956, 35:150-4]. The unesterified fatty acidwas removed through organic extraction using n-heptane. Acyl-CoA formedduring the reaction remained in the aqueous fraction and was quantifiedby scintillation counting. Data were expressed as nmol oleoyl CoAformed/min/mg protein.

Example 7 Assessment of the Trans Epithelial Electrical Resistance(TEER)

Caco-2 cells were seeded at a density of 2.5×10⁵ cells/cm² in a 12-wellsystem using Collagen-Coated Transwell®-COL Inserts (Corning LifeSciences). For growth and differentiation, 100 μL of relevant culturemedium was added to the upper compartment and 600 μL was added to thelower compartment of each transwell. To assess integrity of theepithelial barrier after compound treatment, the trans epithelialelectrical resistance (TEER) test was used. Confluent and fullydifferentiated cells were starved of serum for 1 h in MEM without phenolred. Subsequently, 100 μL of MEM containing the selected compound or MEMalone were added to the upper compartment and cells were furtherincubated 1 h at 37° C., 5% CO₂. Trans epithelial electrical resistancewas then measured using the Millipore Millicell®-ERS system. For eachexperiment, background resistance was determined on a transwell insertwithout cells.

Example 8 Inhibition of Uptake of [³H]-Oleic Acid by Caco-2 Cells afterCompound Treatment

To assess the inhibition of fatty acid transport using a native fattyacid ligand, 5 μM [³H]-oleate (3.75 μmol/μCi) in 5 μM BSA was added to60 mm culture dishes containing monolayers of confluent Caco-2 cellsdifferentiated as described above that had been pretreated with thecompound of interest for 1 h. The transport reaction was initiated bythe addition of [³H]-oleate and incubation was continued for 3 min.Uptake was stopped by the addition of 6 mL of 100 μM BSA prepared inMEM. The stop cocktail was removed by aspiration and cells rinsed oncewith a solution 50 μM BSA in MEM to eliminate non-transported fattyacid. Cells were trypsinized, scraped from the culture disk andcollected by centrifugation (5 min, 1,500×g). The supernatant wasdiscarded and cells were resuspended in 1 mL of MEM. Triplicate aliquots(20 μL) of the cell suspension were used to measure cell-associatedradioactivity, which is reflective of fatty acid transport. A cellaliquot of 40 μL, was used to determine the number of viable cells usinga bright-line hematocytometer in the presence of 0.4% Trypan blue (w/v).Results were expressed as pmol of fatty acid transported/100,000 cells/3min. Experiments were repeated at least 3 times.

Example 9 Assessment of Glucose Uptake into Caco-2 Cells

To assess the impact of selected compounds upon glucose transport,Caco-2 cells were seeded and differentiated in 96-well collagen coatedplates as detailed above and incubated for 1 h with compound diluted inPBS. Glucose transport was assessed by the addition of 50 μL of2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG;final concentration 256 μM) a fluorescent glucose analog [Yamada et al.,J. Biol. Chem., 2000, 275:22278-83]. Cells were incubated 30 min at 37°C., 5% CO₂ to allow uptake. The medium was removed by aspiration andcells were rinsed once with 100 μL of PBS. Cell-associated fluorescencewas measured using a Bio-Tek Synergy HT multidetection microplate readerusing 485±20 nm (excitation), 528±20 nm (emission) filter set.

Example 10 Data Analysis

Values in arbitrary units of the fluorescent signals (AFU) from each HTSplate were acquired using KC4 software in a BioTek Synergy plate reader.These values were exported to Excel (Microsoft Corp., Redmond, Wash.)spreadsheet templates and the assay quality control Z′ factor wascalculated as described [Li et al., J. Lipid Res., 2008, 49:230-44].ChemTree software (Golden Helix, Inc., Bozeman, Mont.) was used foradditional statistical analysis and to study structure-activityrelationships.

Example 11 Results of High Throughput Screening in Humanized Yeast

Using yeast cells in which fatty acid transport was dependent uponhsFATP2, two libraries with over 100,000 chemically diverse compoundswere screened for inhibition of fatty acid transport using thefluorescent fatty acid analogue C₁-BODIPY-C₁₂ [Li et al., J. Lipid Res.,2008, 49:230-44; Li et al., Anal. Biochem., 2005, 336:11-9]. Using aselection criterion of a change in fluorescence of 3 standard deviationunits from the mean of untreated control cells, this screen identified234 compounds as potential inhibitors of fatty acid transport. Of these,8 were eliminated because they quenched the fluorescent signal ofC₁-BODIPY-C₁₂; another 10 were eliminated because they apparentlydisrupted the membrane and increased permeability. The remaining 216compounds were clustered into structural classes using ChemTree (GoldenHelix) and JChem (ChemAxon) analysis software. Compounds that weresimilar in structure to the atypical antipsychotics identified in aprevious screening trial were not considered further since compounds ofthis type may cause hypertriglyceridemia and other metabolicdisturbances upon chronic administration in patients [Li et al., J.Lipid Res., 2008, 49:230-44].

After structural clustering, most compounds fell into 5 structuralclasses and a representative of each was chosen for extensivecharacterization in yeast, 3T3-L1 adipocytes, HepG2 hepatocytes andCaco-2 intestinal epithelial cells. Table 1 lists the names and showsthe structures of the selected compounds, as well as the IC₅₀ fortransport inhibition in Caco-2 and HepG2 cells as well as 3T3-L1adipocytes. These compounds inhibited fatty acid transport in Caco-2 andHepG2 cells at concentrations in the low micromolar range (Table 1) andresulted in sigmoidal dose response curves (FIG. 1). These compoundswere not as effective in inhibiting C1-BODIPY-C12 transport in 3T3-L1adipocytes and resulted in higher IC₅₀ values (Table 1, FIG. 2), whichmay be reflective of the different isoforms of FATP being expressed.When tested in Caco-2 cells, none of the selected compounds resulted incellular toxicity at 10 times the IC₅₀ dosage as measured using the MTTassay (FIG. 3).

TABLE 1 Structure and Activity of Inhibitory Compounds 3T3-L1 Caco-2HepG2 Adipocytes Compound Compound IC₅₀ IC₅₀ IC50 Code ID ChemicalStructure^(a) (μM)^(b) (μM) (μM) I-1 5584680

3.99 ± 0.32 7.55 ± 1.23 231.1 ± 33.5  II-1 5674122

6.34 ± 0.93 10.15 ± 1.26  900.9 ± 204.6 III-1 6022155

4.93 ± 0.40 6.72 ± 0.81 52.5 ± 6.7  IV-1 5675786

6.66 ± 0.64 ND ND V-1  372127

29.22 ± 3.64  84.12 ± 14.12 142.9 ± 22.4  ^(a)Systematic Names: I-1,(5E)-5-[(3-bromo-4-hydroxy-5-methoxyphenyl)methylene]-3-(3-chlorophenyl)-2-thioxothiazolidin-4-one;II-1,2-benzyl-3-(4-chlorophenyl)-5-(4-nitrophenyl)-1H-pyrazolo[5,1-b]pyrimidin-7-one;III-1,2-[7-(trifluoromethyl)-2,3-dihydro-1H-1,4-diazepin-5-yl]naphthalen-1-ol;IV-1,5′-bromo-5-(6-oxocyclohexa-2,4-dien-1-ylidene)spiro[1,3,4-thiadiazolidine-2,3′-1H-indole]-2′-one;and V-1 2,4-bis(1H-indo1-3-yl)butan-1-ol ^(b)Values are the averages of3 experiments plus and minus the standard error of the mean. ND, notdetermined

Example 12 Reversibility of Selected Compound Action on Fatty AcidTransport

Whether or not inhibition of fatty acid transport was reversible wasevaluated by incubating Caco-2 cells with a test compound for one hour,rinsing the cells with MEM and replacing the culture media. After a 24hour recovery period, fatty acid uptake returned to essentially thepretreatment levels (Table 2). Further characterization was aimed atconfirming specific hsFATP mediated fatty acid transport inhibition bythese different compounds.

TABLE 2 Compound Inhibition is Reversible % Transport Standard %Transport after 24 Compound^(a) Assay^(b) Hour Recovery Control (nocompound) 100 100 I-1 46 ± 3 93 ± 13 II-1 54 ± 1 88 ± 18 III-1 35 ± 1 74± 12 IV-1 27 ± 1 99 ± 16 V-1 52 ± 2 99 ± 16 ^(a)All compounds added atf.c. 40 μM to Caco-2 cells ^(b)Numbers are the mean of 3-5 experiments ±the standard deviation

Example 13 Inhibition of Fatty Acid Transport was not Due to NonspecificEffects on the Membrane

It was a concern that treatment with a compound might have adverseeffects on membrane barrier function by altering, for example, membranelipid or protein composition. To test if this might be the case, thetrans epithelial electrical resistance (TEER) was evaluated in Caco-2cells treated with each of the compounds. None of the compounds caused asignificant (p<0.05) change in membrane permeability by this test (FIG.4A).

To evaluate whether other membrane specific processes were disrupted,the transport of the fluorescent glucose analogue, 2-NBDG, intodifferentiated Caco-2 cells was measured following incubation with theselected compounds. Glucose transport was not affected by compounds I-1,II-1, III-1 or IV-1 at 20 μM, a dosage that maximally inhibitsC₁-BODIPY-C₁₂ transport. V-1 at 50 μM reduced 2-NBDG transport slightly(FIG. 4B).

Example 14 Inhibition of the Transport of Native Fatty Acids

The fluorescent fatty acid analogue C₁-BODIPY-C₁₂ is transported andmetabolized in a manner similar to the natural fatty acids [Li et al.,J. Lipid Res., 2008, 49:230-44; Sabah et al., Exp. Eye Res., 2005,80:31-6]. However, there is always the concern the three fused rings ofthe BODIPY moiety that do not occur in natural fatty acids mightinteract with the fatty acid transport apparatus in a manner that isdistinct from the native ligands. Therefore, fatty acid transport intocells was measured using radioactively labeled oleate ([³H]-C_(18:1)).The transport of [³H]-C_(18:1) was linear within the first 5 minutesfollowing initiation of the experiment. To test inhibition by theidentified fatty acid transport inhibitors, differentiated Caco-2 cellswere treated with the selected compounds for 1 hour and measured fattyacid transport using [³H]-C_(18:1) over a 3 minute period. As shown inFIG. 5, the transport of oleate, a representative native fatty acidligand, was significantly inhibited by all 5 compounds (p<0.01). Theseobservations corroborate results obtained using C₁-BODIPY-C₁₂ to monitorfatty acid transport and inhibition.

Example 15 Acsl Activity Remained Unchanged Following Compound Treatment

Upon entry into a cell, fatty acids are activated to CoA thioesters byacyl-CoA synthetase (Acsl) in order to enter lipid metabolic pathways.It has been shown in yeast that both FATP and Acsl are required forfatty acid transport through the process of vectorial acylation[Faergeman et al., J. Biol. Chem., 2001, 276:37051-9; Faergeman et al.,J. Biol. Chem., 1997, 272:8531-8]. As the small compound inhibitors wereselected using a live cell assay, there remained the possibility thatAcsl, as opposed to hsFATP2, was the target, thereby blocking fatty acidtransport. To address whether Acsl was the target of the selectedcompounds, differentiated Caco-2 cells were pretreated with a compoundfor 1 hour, the compound was removed by rinsing, and then oleoyl CoAsynthetase activity was assayed in total cell extracts. Oleoyl CoAsynthetase activity was not reduced by treatment with II-1, III-1, IV-1,or V-1 (FIG. 6). I-1, on the other hand, at 50 μM (10 times the IC₅₀),inhibited activity by about 50%. Similar results were obtained when thecompounds were added to cell extracts rather than whole cells.

Example 16 Comparison of the Activities of Structurally RelatedCompounds

To test structural features important in inhibition of fatty acidtransport by the selected compounds, fatty acid transport inhibition ofrelated compounds available from the ChemBridge compound database wasnext addressed. The structures can be found in Table 3, and thepredicted chemical properties can be found in Table 4. Five compoundssimilar to I-1 were evaluated, and most were 50 to 100 times less potentthan the parent compound (Table 3). One compound, I-4, had an IC₅₀similar to I-1. The only difference between I-1 and I-4 is that I-4contains a fluorine on the R1 phenolic ring while I-1 contains achlorine in that position. Replacing the sulfur with oxygen at positionR2 increases the IC₅₀ by 25- to 126-fold. The positions of the methylether, hydroxyl and Br on the R3 phenolic ring in I-1 and I-4 areidentical and likely contribute to the ability to block fatty acidtransport with comparable efficiency. Three related compounds similar toII-1 were tested and it was found that the nitrite at position R1appears to have an influence on activity at therapeutic levels (Table3B). Only one compound similar to III-1, III-2, was tested, and it wasnearly 50-fold less effective (Table 3C). The R groups differconsiderably between III-1 and III-2 and, thus, fewer conclusions can bemade regarding functional groups that disrupt fatty acid transport. Twoadditional compounds related to IV-1 were tested next and it was foundthat both had IC₅₀s in the same range as IV-1 (Table 3D). Thus, thebromine at position R2 appears to be more important to impart activityas opposed to the hydroxyl at position R1.

TABLE 3 Analysis of compounds structurally related to I-1, II-1, III-1and IV-1 in Caco-2 cells A. Formula (I) compounds

Com- IC₅₀ pound R1 R2 R3 (μM) I-1

S

3.99 ± 0.32 I-2

S

121 ± 25  I-3

S

468 ± 212 I-4

S

4.7 ± 0.8 I-5

O

504 ± 340 I-6

O

102 ± 26  B. Formula (II) compounds

IC₅₀ Compound R1 R2 (μM) II-1 NO₂ Cl 6.34 ± 0.93 II-2 NO₂ OCH₃ 17.3 ±3.01 II-3 NO₂ H 16.4 ± 2.22 II-4 H CL 76.9 ± 15.7 C. Formula (III)compounds

IC₅₀ Compound R (μM) III-1

4.93 ± 0.40 III-2

241 ± 57  D. Formula (IV) compounds

IC₅₀ Compound R1 R2 (μM) IV-1 OH Br 6.66 ± 0.64 IV-2 OH H 11.9 ± 1.43IV-3 H Br 4.84 ± 0.69

TABLE 4 Chemical properties of selected compounds Compound IC₅₀ tPSA H HRule of 5 Name/ID MW (μM ± SE) logP RB (Å) donor acceptor violationsI-1/5584680 457 3.99 ± 0.32 5.45 2 96.2 4 4 1 I-2/5361510 378  121 ±25.4 4.28 4 96.2 1 4 0 I-3/5582711 359 468 ± 212 3.38 5 105.4 2 5 0I-4/5674481 440  4.7 ± 0.82 3.39 4 96.2 1 4 0 I-5/6760222 441 504 ± 3404.85 4 81.1 1 5 0 I-6/7140230 441  102 ± 26.1 4.85 4 81.1 1 5 0II-1/5674122 457 6.34 ± 0.93 5.7 4 87.8 1 5 1 II-2/5670982 452 17.3 ±3.01 5.0 5 97.1 1 6 0 II-3/5675245 422 16.4 ± 2.22 5.0 4 87.8 1 5 0II-4/5679381 412 76.9 ± 15.7 5.8 4 44.7 1 3 1 III-1/6022155 306 4.93 ±0.40 2.91 2 44.6 2 3 0 III-2/6001852 278  241 ± 57.01 3.4 NA NA 2 6 0IV-1/5675786 376 6.66 ± 0.64 4.15 0 69.2 3 5 0 IV-2/5681753 297 11.9 ±1.43 1.19 1 73.7 3 3 0 IV-3/5830995 360 4.84 ± 0.69 2.97 1 53.5 2 2 0Data for IC50 derived from laboratory data. All additional informationfrom PubChem and ChemSpider databases; RB, rotatable bonds; tPSA,topological polar surface area; NA, not available

Example 17 Animal Experiments

C57BL/6 female mice (age 8 weeks) are placed on a 60% high fat (lard)diet (TestDiet formula 58G9) for 4 weeks prior to compoundadministration to allow time for the animals to adjust to the diet,begin to gain weight, and to elevate serum lipid levels. A second dietwith the same composition but 12% fat calories is used as a control(TestDiet formula 58G7). The normal fat and calorie diet is alsonecessary to identify alterations in eating behavior, weight loss orgain, and serum lipid levels apart from the high fat diet regime. Thecompounds being tested are administered either by gavage or IP injection(each dosage at 5 or 100 mg/kg) once per day for 7 days. The lipaseinhibitor, Orlistat (Xenical™), is administered at the same dosage forcomparison since this compound is known to inhibit fat absorption. Micehave free access to food and water over the course of the study.Controls include mice receiving vehicle alone and maintained on eachdiet. Experiments use 3-12 mice per group.

Example 18 Assessment of Compound Treatment on Growth and Overall Health

To evaluate effects due to treatment with the compound, mice areobserved visually once per day to verify normal activities andbehaviors. Weight is recorded once per week during the preliminaryfeeding period and then every day once administration of the compoundhas begun. Food intake is measured periodically during the 4 weekpretrial period and daily during the 7 day compound treatment schedule.

Example 19 Measurement of Blood Lipids and Serum Chemistries

Serum lipids are measured using blood samples (obtained from periorbitalbleeding of anesthetized animals) on the day the high fat diets arebegun, at week 2 and week 5 prior to administration of the first dose ofcompound, and then again on the last (7^(th)) day of compound treatment.Lipids are extracted from serum samples using Folch extraction method(Folch et al., J. Biol. Chem., 1957, 226:497-509). Triglycerides andcholesterol levels are measured using commercial kits (Wako).

To assess compound effects on liver function, whole blood is drawn bycardiac puncture at the termination of the experiment (50-100 μl) andanalyzed using the Mammalian Liver Profile rotor in a VetScan analyzer(Abaxis). This provides data for alanine transaminase (ALT), alkalinephosphatase (ALP), blood urea nitrogen (BUN), albumin (ALB), bile acids(BA), total cholesterol, GGT, and total bilirubin (TBIL).

Example 20 Histological Analysis of Tissues and Measurement ofTriglyceride Accumulation

To assess whether or not the compounds limit fat deposition in tissues,particularly liver, lipid droplet accumulation is evaluated (Sealls etal., Biochim. Biophys. Acta, 2008, 1781:406-14). C57BL/6 mice inparticular are prone to obesity-associated fatty liver and it isexpected that the control mice (receiving vehicle alone) on the high fatdiet will present this phenotype. For these experiments, at the end ofthe 7 day compound treatment, animals are sacrificed and necropsiesperformed. Tissue samples (e.g., liver, soleus muscle, heart, pancreasand adipose) are stored frozen at −80° C. and includes untreatedtissues, portions treated with RNAlater® for RNA extraction, portionstreated with OCT then snap frozen for Oil red O, and formalin-treatedtissues imbedded in paraffin blocks for histology. Sectioning andimaging of tissues is performed by the UNL Center for BiotechnologyMicroscopy Core Facility. Lipid extraction, quantification and analysisare carried out by standard laboratory procedures.

Example 21 Measurement of Sterol and Fatty Acid Content of Feces

Sterol and fatty acid absorption are studied with respect to compoundtreatment. For fecal analysis of lipids, animals are individually housedand food and water provided ad libidum. Early in the morning, bedding ischanged and then feces are collected after six hours. These experimentsare performed prior to initiation of the diets, prior to drug treatmentduring the 4^(th) week of the diets, and on days 5-7 of compoundtreatment. Lipids are extracted from fecal samples using Folchextraction methods. Nonadecanoic acid and 5α-cholestane are added astracers for recovery of fatty acids and sterols, respectively. Thesamples are split for identification and quantitation of sterols,oxysterols, and fatty acids using routine GC-MS. Bile acids are measuredby enzymatic assay employing 3α-hydroxysteroid dehydrogenase. Totaltriglycerides are measured using a commercially available kit (Wako).Lipid mass is normalized to food consumption and dietary fat content fordetermination of percent fat absorption.

Example 22 Rate of Uptake of Fatty Acids

Fecal lipid content provides a crude estimate of lipid absorption due todiet and/or compound treatment. To more carefully assess fatty acidabsorption, an isotopic tracer method is used. For these experiments,mice are fasted overnight on day 7 and intravenously injected with 500mg/kg Tyloxapol to block serum lipase activity and to allow serum lipidaccumulation to be monitored. Mice are gavaged with 500 μl of anIntralipid Fat Emulsion mixture containing 10 μCi [³H]-triolein. Bloodsamples (obtained via tail clip) are collected at time zero and everyhour after gavage for 4 hours. Mice are sacrificed after the finalbleed, and serum radioactivity and triglyceride content determined byscintillation counting of each sample.

Example 23 Uptake of Cholesterol

The fatty acid uptake inhibitors are expected to be specific for fattyacids and are not expected to effect uptake of sterols. To verify thisoutcome, the fecal dual isotope method is used to assess sterolabsorption. Mice fed ad libidum with the high fat or control diets andthen treated with the test compound or vehicle will be gavaged on twoconsecutive days (days 4 and 5 of compound treatment) with 1 μCi [¹⁴C]cholesterol and 2 μCi [³H] sitosterol. Sitosterol is used as anon-absorbed control sterol. Fecal samples from each mouse are collectedfor 3 days after the initial dose (days 5, 6 and 7). Lipids areextracted by the standard Folch procedure and then radioactivity isdetermined by scintillation counting.

Cholesterol uptake is determined as:

% uptake=(¹⁴C/³H gavage−¹⁴C/³H fecal)/(¹⁴C/³H gavage)−100%.

It is to be understood that, while the methods and compositions ofmatter have been described herein in conjunction with a number ofdifferent aspects, the foregoing description of the various aspects isintended to illustrate and not limit the scope of the methods andcompositions of matter. Other aspects, advantages, and modifications arewithin the scope of the following claims.

Disclosed are methods and compositions that can be used for, can be usedin conjunction with, can be used in preparation for, or are products ofthe disclosed methods and compositions. These and other materials aredisclosed herein, and it is understood that combinations, subsets,interactions, groups, etc. of these methods and compositions aredisclosed. That is, while specific reference to each various individualand collective combinations and permutations of these compositions andmethods may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a particularcomposition of matter or a particular method is disclosed and discussedand a number of compositions or methods are discussed, each and everycombination and permutation of the compositions and the methods arespecifically contemplated unless specifically indicated to the contrary.Likewise, any subset or combination of these is also specificallycontemplated and disclosed.

1-75. (canceled)
 76. A method of inhibiting fatty acid uptake by cells,comprising: contacting said cells with a compound having the formula:

or a pharmaceutically acceptable salt form thereof, wherein: R¹ is H orNO₂; and R² is selected from the group consisting of: H, Cl, and OCH₃.77. The method of claim 76, wherein said cells are selected from thegroup consisting of intestinal epithelial cells and hepatocytes.
 78. Themethod of claim 76, wherein said contacting is in vitro.
 79. The methodof claim 76, wherein said inhibition is reversible.
 80. The method ofclaim 76, wherein R¹ is NO₂.
 81. The method of claim 76, wherein R² isCl.
 82. The method of claim 76, wherein the compound is selected fromthe group consisting of:

or a pharmaceutically acceptable salt form thereof.
 83. The method ofclaim 76, wherein the compound is:

or a pharmaceutically acceptable salt form thereof.
 84. A method oftreating a disease associated with dyslipidemias and lipotoxicity,comprising: administering, to an individual, a compound having theformula:

or a pharmaceutically acceptable salt form thereof, wherein: R¹ is H orNO₂; and R² is selected from the group consisting of: H, Cl, and OCH₃.85. The method of claim 84, wherein said disease is selected from thegroup consisting of obesity, metabolic syndrome, insulin resistantdiabetes, cardiovascular disease, and non-alcoholic fatty liver disease.86. The method of claim 84, wherein R¹ is NO₂.
 87. The method of claim84, wherein R² is Cl.
 88. The method of claim 84, wherein the compoundis selected from the group consisting of:

or a pharmaceutically acceptable salt form thereof.
 89. The method ofclaim 84, wherein the compound is:

or a pharmaceutically acceptable salt form thereof.
 90. A method ofinhibiting FATP2 in a cell, the method comprising: contacting said cellwith a compound having the formula:

or a pharmaceutically acceptable salt form thereof, wherein: R¹ is H orNO₂; and R² is selected from the group consisting of: H, Cl, and OCH₃.91. The method of claim 90, wherein said cell is selected from the groupconsisting of intestinal epithelial cells and hepatocytes.
 92. Themethod of claim 90, wherein said contacting is in vitro.
 93. The methodof claim 90, wherein said inhibition is reversible.
 94. The method ofclaim 90, wherein R¹ is NO₂.
 95. The method of claim 90, wherein R² isCl.
 96. The method of claim 90, wherein the compound is selected fromthe group consisting of:

or a pharmaceutically acceptable salt form thereof.
 97. The method ofclaim 90, wherein the compound is:

or a pharmaceutically acceptable salt form thereof.